Notes

Abstract:

Drugs in the amphetamine class, 3,4-methylenedioxymethamphetamine (MDMA), and amphetamine (AMPH) are among the most popular drugs of abuse. Chronic users of amphetamine or analogs can have long-lasting deficits in dopaminergic or serotonergic systems. Planaria, thought to possess the first true central nervous system, exhibit pharmacological and behavioral similarities to vertebrates for many serotonin, dopamine, acetylcholine, GABA, glutamate, and endorphin receptors and drugs, have regenerative capabilities, and exhibit an anti-tropism to light. This study evaluated the toxic effects of AMPH and MDMA on the planarian species D. dorotocephala. The first set of experiments focused on evaluating general toxic effects induced by these drugs. We found that AMPH and MDMA dose-dependently caused decreased locomotor speed and head loss. These effects were not prevented by treatment with drugs shown to be neuroprotective in some other models. Both also caused depletion of monoamine levels and increased indices of dopamine turnover, an effect observed in many vertebrate models and indicative of toxicity. The next set of experiments sought to determine the long-term effects from head loss resulting from AMPH or MDMA exposure. We found that following head loss in AMPH, but not MDMA, planaria did not recover locomotor speed to control levels. The final set of experiments evaluated the effects of low doses of AMPH and MDMA on regeneration following head removal. We found that low doses of either drug did not inhibit regeneration. Our studies showed that both AMPH and MDMA are toxic to planaria and appear to be affecting the monoaminergic systems in a way similar to what is observed in other vertebrate models. This model could be useful for studying the dopamine and serotonin related toxicity resulting from abuse of amphetamines.

General Note:

In the series University of Florida Digital Collections.

General Note:

Includes vita.

Bibliography:

Includes bibliographical references.

Source of Description:

Description based on online resource; title from PDF title page.

Source of Description:

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

Thesis:

Thesis (Ph.D.)--University of Florida, 2008.

Local:

Adviser: Baxter Jr, Lewis Rhea.

Local:

Co-adviser: Gold, Mark S.

Electronic Access:

RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-11-30

Notes

Abstract:

Drugs in the amphetamine class, 3,4-methylenedioxymethamphetamine (MDMA), and amphetamine (AMPH) are among the most popular drugs of abuse. Chronic users of amphetamine or analogs can have long-lasting deficits in dopaminergic or serotonergic systems. Planaria, thought to possess the first true central nervous system, exhibit pharmacological and behavioral similarities to vertebrates for many serotonin, dopamine, acetylcholine, GABA, glutamate, and endorphin receptors and drugs, have regenerative capabilities, and exhibit an anti-tropism to light. This study evaluated the toxic effects of AMPH and MDMA on the planarian species D. dorotocephala. The first set of experiments focused on evaluating general toxic effects induced by these drugs. We found that AMPH and MDMA dose-dependently caused decreased locomotor speed and head loss. These effects were not prevented by treatment with drugs shown to be neuroprotective in some other models. Both also caused depletion of monoamine levels and increased indices of dopamine turnover, an effect observed in many vertebrate models and indicative of toxicity. The next set of experiments sought to determine the long-term effects from head loss resulting from AMPH or MDMA exposure. We found that following head loss in AMPH, but not MDMA, planaria did not recover locomotor speed to control levels. The final set of experiments evaluated the effects of low doses of AMPH and MDMA on regeneration following head removal. We found that low doses of either drug did not inhibit regeneration. Our studies showed that both AMPH and MDMA are toxic to planaria and appear to be affecting the monoaminergic systems in a way similar to what is observed in other vertebrate models. This model could be useful for studying the dopamine and serotonin related toxicity resulting from abuse of amphetamines.

General Note:

In the series University of Florida Digital Collections.

General Note:

Includes vita.

Bibliography:

Includes bibliographical references.

Source of Description:

Description based on online resource; title from PDF title page.

Source of Description:

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

Thesis:

Thesis (Ph.D.)--University of Florida, 2008.

Local:

Adviser: Baxter Jr, Lewis Rhea.

Local:

Co-adviser: Gold, Mark S.

Electronic Access:

RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-11-30

Record Information

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UFRGP

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Applicable rights reserved.

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lcc - LD1780 2008

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UFE0022137:00001

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1 CHARACTERIZATION OF THE EFFECTS OF AMPHETAMINES IN THE PLANARIAN SPECIES Dugesia dorotocephala By SHANNON JANSSEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Shannon Janssen

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3 To my mom, dad, and brother, whose enc ouragement and support have been essential

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4 ACKNOWLEDGMENTS There are many people who have been involved with me through my graduate experience. They have all helped me in various ways. T hose who had the largest impacts deserve to be recognized. I would like to thank my advisors Lewis Baxter and Mark Gold and my committee members Mark Lewis, Mike Ki ng, and Joanna Peris for their guidance and support. This has been a long road and they have all been integral to the process. I am grateful for the endless support and assistance they have provided. I would especially lik e to thank Dr. Baxter and Dr. Gold for continuing to believe in me. Dr. Sue Semple-Rowland and BJ Streetman have particularly provided much support during the past year and it is very much apprec iated. My family has also been an incredible source of support for me and I would not be here today without them. Lastly, I would like to thank all my friends who have stood by me.

8 LIST OF FIGURES Figure page 3-1 Test apparatus used to determine locomoto r speed of anti-tropism to a standard light gradient....................................................................................................................... .......39 3-2 Location of head removal..................................................................................................41 4-1 Head loss resulting from amphetamine exposure..............................................................49 4-2 Death resulting from amphetamine exposure....................................................................50 4-3 Locomotor speed following amphetamine treatment........................................................51 4-4 Dose-dependent head loss resu lting from amphetamine exposure....................................52 4-5 Intermediate doses of ampheta mine result in minimal death.............................................53 4-6 Dose-dependent decrease in locomotor speed following treatment with amphetamine...54 4-7 Head loss following MDMA administration. ..................................................................55 4-8 Locomotor speed following MDMA administration.........................................................56 4-9 Representative photograph of degenerati on in head region following exposure to AMPH........................................................................................................................... .....57 4-10 Representative photograph of degenerati on in head region following exposure to MDMA........................................................................................................................... ....58 4-11 Changes in levels of 5-HT following treatment with AMPH and MDMA.......................59 4-12 Changes in levels of DA following treatment with AMPH and MDMA..........................60 4-13 Changes in levels of DOPAC/DA follow ing treatment with AMPH and MDMA. ..........61 4-14 Head loss following amphetamine exposure re sults in long-term locomotor deficits.......62 4-15 Representative photographs of head morphology after AMPH.........................................62 4-16 Head loss following MDMA exposure doe s not result in long-term locomotor deficits....................................................................................................................... .........63 4-17 Differential effects of losing heads in AMPH vs. being mechanically removed...............64 4-18 Dose-dependent head loss resulting from DOI exposure..................................................65 4-19 Death resulting from DOI..................................................................................................66

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9 4-20 DOI dose-dependently results in a decrease in locomotor speed......................................67 4-21 Methiothepin potently effects locomotor speed.................................................................68 4-22 Amphetamine did not in hibit head regeneration................................................................70 4-23 MDMA did not inhibit head regeneration.........................................................................71

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10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF THE EFFECTS OF AMPHETAMINES IN THE PLANARIAN SPECIES Dugesia dorotocephala By Shannon Janssen May 2008 Chair: Lewis Baxter Cochair: Mark Gold Major: Medical SciencesNeuroscience Drugs in the amphetamine class, 3,4-methylenedioxymethamphetamine (MDMA), and amphetamine (AMPH) are among the most popu lar drugs of abuse. Chronic users of amphetamine or analogs can have long-lasting defi cits in dopaminergic or serotonergic systems. Planaria, thought to possess the first true centr al nervous system, exhibit pharmacological and behavioral similarities to vertebrates fo r many serotonin, dopamine, acetylcholine, GABA, glutamate, and endorphin receptors and drugs, have regenerative capabilities, and exhibit an antitropism to light. This study evaluated the toxi c effects of AMPH and MDMA on the planarian species D. dorotocephala The first set of experiments focused on eval uating general toxic e ffects induced by these drugs. We found that AMPH and MDMA dose-de pendently caused decreased locomotor speed and head loss. These effects were not preven ted by treatment with drugs shown to be neuroprotective in some other models. Both al so caused depletion of monoamine levels and increased indices of dopamine turnover, an e ffect observed in many vertebrate models and indicative of toxicity. The next set of experiments sought to determine the long-term effects from head loss resulting from AMPH or MDMA expos ure. We found that following head loss in

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11 AMPH, but not MDMA, planaria did not recover lo comotor speed to control levels. The final set of experiments evaluated the effects of lo w doses of AMPH and MDMA on regeneration following head removal. We found that low doses of either drug did not inhibit regeneration. Our studies showed that both AMPH and MDMA are toxic to planaria and appear to be affecting the monoaminergic systems in a way sim ilar to what is observed in other vertebrate models. This model could be useful for studyi ng the dopamine and seroto nin related toxicity resulting from abuse of amphetamines.

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12 CHAPTER 1 AMPHETAMINES Epidemiology Drug abuse is a major health problem throughout the world. The estimated costs of drug abuse, including loss of productivity, crime, a nd other health-related expenses for the United States alone was estimated at over $140 billion in 1998 (NIDA, 2001). This was an increase by $40 billion from 1992, and such use is projected to continue increasing accord ing to the Office of National Drug Control Policy. One particular gr oup of abused drugs, the amphetamines, has gained in popularity especially throughout California a nd the midwest and is spreading to other areas throughout the country as well. Accordi ng to the Drug Abuse Warning Network (DAWN), stimulants, including all amphetamines, but particularly amphetamine (AMPH) and methamphetamine (METH), were involved in ov er 100,000 Emergency Department (ED) visits, accounting for about 5% of drug-related ED vi sits overall in 2004 (SAMHSA, 2006a). Although stimulant abuse is not as high now as it was in mid 1900s, these remain heavily abused among illicit drug users. With accumulating evidence of the lasting effects on the dopaminergic systems of chronic amphetamine abusers in humans, th is poses a serious and urgent need for an understanding of the mechanisms involved in AMPH toxicity. AMPH first came into widespread use during the 1950s and s when it was frequently prescribed for medical purposes such as: as a br onchodilator in the form of an inhaler, as an anorectic to promote weight loss, and as a treatment for narcolepsy. It was even given to soldiers as a stimulant to combat fatigue during duty. Th is raised general awar eness about the drug and its euphoric effects, which subsequently catalyzed the epidemic of illicit recreational use. Laws were then passed in the U.S. and many other countr ies, providing sanctions in order to counteract the epidemic and limit the legal production of AMPH. It is still prescribed, in low doses, to treat

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13 attention deficit disorder, narc olepsy, and obesity. Recreational use of AMPH, however, remains popular and the substituted form METH, which ha s similar effects, has especially gained popularity. AMPH for abuse can be obtained through prescriptions so ld illegally or it can be synthesized in clandestine laborator ies. It can be ingested orally or injected intravenously (or smoked in the case of METH). The use pattern of smoking represents the most hazardous form for ingesting METH. Another popular drug, 3,4-methylenedioxymet hamphetamine, MDMA (ecstasy), a ringsubstituted amphetamine and a club drug, is popular among young people, especially in the dance club and rave scene in the U.S., the United Kingdom, and Europe. According to the monitoring the future survey published by the National Institute on Drug Abuse, lifetime ecstasy use in the U.S. decreased between 2002 and 2005 (from 3.3% to 1.6%) in the 12-17 age group and among 18-25 year olds (15.1% in 2002 to 13.7% in 2005) (SAMHSA, 2006b). However, according to the Office of National Dr ug Control Policy (ONCDP), the number of new users increased from 2005 to 2006 and the number of past month users also increased in the same time period (ONCDP, 2008). MDMA was in itially synthesized in 1912 by Merck, but it was not produced and did not come into use until the 1970s and s where it was used to aid in psychotherapy. It was reported to facilitate ope n communication and an increased sense of well being in patients (Grinspoon and Bakalar, 1986). It then gained popularity with recreational users for its mood-enhancing effects and the food and drug administration (FDA) classified it as a Schedule I drug in 1985, thus eliminating any lega l use in this country. There is currently a growing movement to reevaluate this classificat ion and make allowances for its limited use in some psychiatric conditions (such as in the trea tment of post-traumatic stress disorder) and also

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14 to aid terminal cancer patients by enhancing th eir mood and perceived quality of life as they approach their impending death. Mechanism of Action AMPH and MDMA exert their effects by faci litating release of catecholamines and serotonin through the transporters and blocking reuptake at presynaptic terminals (Rudnick and Wall, 1992; Sulzer et al., 1995; Mlinar and Corradetti, 2003). AMPH exerts its effects primarily through the dopamine transporter (DAT). It en ters the nerve terminal by two mechanisms: passive diffusion and through the transporter at the nerve terminal (Zaczek et al., 1991). MDMA acts by similar mechanisms predominantly at the serotonin transporter (SERT) at least in some but not all mammals (Rudnick and Wall, 1992). It should be noted, however, that in some species MDMA has different effects: in some mice models, MDMA acts primarily at the dopaminergic system (Easton and Marsden, 2006), wh ile in rats and non-human primates it acts primarily at the serotonergic system (Rudni ck and Wall, 1992). Both also act at the norepinephrine transporter (NET), though to a lesser extent. Vesicular monoamine transporter 2 (VMAT 2) is also involved in vesicular releas e which is calciumand impulse-dependent. In mice lacking the DAT (KO mice), AMPH fails to cause an increase of DA release in the striatum (in the extracellular space), but it does not inhibit its vesicula r releasing action (Jones et al., 1998). Effect on Monoamine Levels Both AMPH and MDMA induce lasting depletions in monoamine levels in animal models. While METH appears to affect both the dopamine rgic and the serotone rgic systems, AMPH primarily affects the DA system and MDMA primarily affects the serotonergic system. AMPH, and the substituted form METH, exert actions as i ndirect agonists of DA receptors, as reuptake blockers through the transporters at the syna pse (Heikkila et al., 1975; Han and Gu, 2006) and

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15 also as inhibitors of monoamin e oxidase (Kita et al., 2000). Taken together, these act ions initially lead to an accumulation of DA in the synapse followed by lasting dopamine depletions (Wagner et al., 1980; Ali et al., 1994). MD MA has similar effects on the serotonin system by releasing serotonin into the synaptic space, causing releas e from synaptic vesicles within the terminal, binding to the reuptake site and effectively blocking reuptake while facilitating uptake of itself into the nerve terminal (Rudnick and Wall, 1992). One method both AMPH and MDMA use to release monoamines from the tr ansporter is through exchange, in which AMPH or MDMA get taken up by the DAT or SERT, respectively, which then binds the respective neurotransmitter (DA or 5-HT) and subsequently carries it into th e extracellular space, in what has been termed the exchange diffusion model (Fischer and C ho, 1979). AMPH further prevents DA reuptake by facilitating movement of the DAT away from th e cell surface, lowering the capacity for reuptake (Kahlig et al., 2006). MDMA has also been shown to inhibit synaptosomal and vesicular uptake of both serotonin and dopamine (Bogen et al., 2003). Neurotoxicity Administration of multiple high doses of AMPH to mice resulted in decreased DAT binding in the striatum, indicati ng nerve terminal destruction (S cheffel et al., 1996). Continuous administration of high doses of METH to rats re sulted in toxicity to both DA and 5-HT terminals (Ricaurte et al., 1980; Armstrong and Noguchi, 2004). Nerve terminal loss is evidenced by a loss of DAT, SERT, and VMAT 2 on the membrane surface (Guilarte et al., 2003). This reduced presence could also be due to an adaptive downregulation of the membrane proteins, as some pharmacological agents have been shown to alter DAT and SET levels (Gordon et al., 1996; Zhou et al., 1996). However, VMAT 2 does not app ear to be as sensit ive (Wilson et al., 1996), indicating that a decrease in leve ls of VMAT 2 in combination with decreases in DAT or SERT represents terminal damage ra ther than down-regula tion at the membrane surface. Following a

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16 prolonged abstinence period, there wa s a significant rec overy of dopamine function (Friedman et al., 1998; Cass and Manning, 1999). However, these re sults should be interp reted cautiously as they are based upon the acute toxic dose model rath er than the chronic use pattern seen in many METH and AMPH abusers. High doses of amphetam ines have also been shown to cause lasting depletions of 5-HT in the striatum, hippocampus and frontal cortex (R icuarte et al., 1980; Friedman et al., 1998). There are also lasting depl etions in tyrosine hydr oxylase (Bowyer et al., 1998) and tryptophan hydroxylase activ ities (Che et al., 1995). Ther e are numerous theories of how amphetamines cause damage, including form ation of ROS and oxidative stress, reactive nitrogen species (RNS), mitochondrial disruptio n, and glutamate-mediated excitotoxicity (Fleckenstein et al., 2007). One group has gone so far as to postulate that it is the neurotransmitter-releasing action of amphetamines (due to their ab ility to facilitate monoamine transport outward through the transporter) on DA and 5-HT that is resp onsible for the neuronal damage and that simply acting as a transport bloc ker is not only inadequate to cause damage, but can also have protective effects (Kita et al, 2003 ). DA may partially mediate the 5-HT depletions induced by MDMA since depletion of DA stores with reserpine attenuates damage, but this may be due to thermoregulation since reserpine c ounteracts the hyperthermic effect observed following MDMA administration (Yuan et al., 20 02). This hyperthermic effect has been implicated in the neurotoxicity observed with MDMA in animal models (Malberg and Seiden, 1998). In addition, depletion of DA prior to MDMA administration also attenuates long-term damage (Brodkin et al., 1993). In mice deficient of MAO-B, t oxicity to 5-HT neurons was attenuated (Fornai et al., 2001) This is thought to be due to the uptake of MDMA-induced DA release into 5-HT neurons, where DA is subseq uently metabolized by MAO-B, a process which was already described to produce reac tive products (Sprague et al., 1998).

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17 It was originally thought that amphetamines were only toxic to nerve terminals; that view, however, is currently under fire Cadet and his colleagues have repeatedly shown evidence of apoptotic cell bodies in mouse brains following administration of METH and AMPH (Deng et al., 2002a; Jayanthi et al., 2004; Krasnova et al., 2005). They and others have provided further evidence that AMPH and METH can cause an up -regulation of pro-apoptotic genes and downregulation of anti-apoptotic genes, possibly due to the generation of reactive oxygen species (ROS), and induce apoptosis directly (Stumm et al., 1999; Thiriet et al., 2001; Deng et al., 2002b). There is also evidence in animal models showing that acute exposure of rat brain to MDMA selectively destroys neur ites of serotonergic neurons in brain regions such as the hippocampus and cerebral cortex (Ricaurte et al., 2000; Meyer and Ali, 2002). MDMA is also known to induce apoptosis in human serotonergic choriocarcinoma (JAR) cells (Simantov and Tauber, 1997) and in rat neocortical neurons in culture. Thus, MDMA might induce neurite degeneration as well as apoptotic death in serotonergic systems. There is also evidence that oxidative stress plays a role in MDMA-induced neur otoxicity. Lipid peroxi dation, an indicator of free radical damage, was increased following MD MA administration (Sprague and Nichols, 1995; Colado et al., 1997). Reactive Oxygen Species Oxidative stress has been implicated in ag ing and a number of degenerative diseases including, but not limited to, Alzheimers (Sompol et al., 2008), where it is thought to play a pivotal role, Parkinsons (Chin et al., 2008), and amyotrophic latera l sclerosis (ALS) (Rakhit et al., 2004). Generation of ROS occurs naturally through cellular metabo lism and mitochondrial production of ATP. These basal levels are ke pt low by endogenous antioxidants and enzymes such as superoxide and glut athione peroxidase. There is strong support for the ROS and oxidative stress hypothesis of amphetamines toxicity with two main theories. One theory is that

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18 following the outward flood of DA into the extra cellular space, it auto-oxidizes to 6-hydroxy (OH) DA, producing hydrogen peroxi de in the process (Kita et al 2003). There is also evidence for the involvement of oxygen-based free radical s given that over-express ion of the copper-zinc superoxide dismutase gene in transgenic mice provides protection from the toxic effects of METH (Deng and Cadet, 2000). In addition, administ ration of antioxidants such as ascorbic acid (Wagner et al, 1986), L-carnitine (Virmani et al ., 2003), and N-acetyl-L-cysteine (Hashimoto et al., 2004) protect against METH-induced dama ge. Similarly, N-acetyl-L-cyteine and -phenylN-tert-butyl nitrone, another free radical scavenger, protect against the lipid peroxidation, hydroxyl radical formation, and DA depleti on caused by AMPH (Wan et al., 2005). The second major theory involves the acc umulation of oxidizable dopamine through redistribution from synaptic vesi cles into cytoplasmic compartments, ultimately resulting in terminal injury. In support of this, METH t oxicity was increased in vesicular monoamine transporter 2 (VMAT 2) knockout mice (Fumagalli et al., 1999). Generally there is a resulting depletion in antioxidants and enzymes necessary for antioxidant reactions after toxi c AMPH or MDMA exposure. The thought is that amphetamines increase the level of re active species beyond the capabilities of the antioxidants present and also through the excess DA released. It is thought that the ex cess DA released then autooxidizes and produces DA-quinones, hydroxyl radicals, super oxide radicals, and hydrogen peroxide both intraand extracellularly. H ydrogen peroxide is also formed when MAO deaminates DA and thus, with all the extra DA released, adds to the load for antioxidants. Reactive Nitrogen Species Meanwhile other groups have focused on a role for glutamate-mediated excitotoxicity. AMPH and METH administration re sults in an increase in glutamate release in the ventral

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19 tegmental area (VTA) (Wolf et al., 2000; Mark et al., 2004) and is prevented by both MK-801, an NMDA antagonist, and SCH 23390, a dopamine D1 receptor antagonist administered prior to AMPH (Wolf and Xue, 1999). Additionally, blocka de of the metabotropic glutamate receptor mGlu5 protects against METH toxi city (Battaglia et al., 2002a). Inhibition of neuronal nitric oxide synthase, nNOS, or knockout of the gene en coding nNOS has also been shown to protect against toxicity following amphetamine treatme nt (Imam et al., 2001; Bashkatova et al., 2004). nNOS generates nitric oxide (NO) which can then combine with superoxide radicals (which are increased following AMPH treatment) to form pe roxynitrite, which in tu rn can then induce DNA strand breaks, lipid oxidation, pr otein oxidation and nitration (Koppe nol et al., 1992). The role for glutamate or the nitrergic system in MDMA induced toxicity is less clear. Evidence suggests that the nitrergic system plays a role in DA toxici ty, but not for serotonin toxicity (Itzhak et al., 2004). Mitochondrial Dysfunction There is some evidence that amphetamines inhibit complexes in the electron transport chain (Burrows et al., 2000). However, it is more likely the combination of both ROS and RNS leads to mitochondrial failure and cell death. This mitochondrial disruption leads to a decrease in ATP production (Wan et al., 1999), while there is generally an increase in energy demands (Virmani et al., 2002). Ad ministration of substrates for the electron trans port chain can attenuate toxicity from amphetamines (Stephans et al., 1 998), providing support for the involvement of the mitochondria. While there is evidence of a poptotic cell death (Krasnova et al., 2005), characterized by membrane blebbi ng, activation of apoptotic ge nes, and compartmentalization, there are more nerve terminals that are affecte d, indicating that necrosis plays a larger role, possibly due to the lack of ATP availabl e to activate the apoptotic cascade.

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20 Behavioral Effects in Animals Both AMPH and MDMA have been shown to result in behavioral changes, such as psychosis, impaired memory, aggre ssiveness, impulsivity, and others in humans. These effects have been tied to nerve terminal damage as well as cell death and plastic restructuring (hyperexcitablility etc). In a recent study involving administration of MDMA to mice at levels seen in human users, oxidative stress in the hippocampal region was repo rted (Frenzilli et al., 2007). In addition, they observed hyperexcitability and a reduced threshold for seizure in the hippocampus. Animal studies also show impair ments in cognitive function. Object recognition memory impairments were shown after both acute (Bisagno et al., 2003) and repeated (Kamei et al., 2006) METH exposure. Neonatal METH expos ure causes impaired hippocampus-dependent spatial memory in rodents in the Morris water maze (Vorhees et a., 2000; Acevedo et al., 2007) and the Barnes maze (Williams et al., 2003). In addition, neurodegeneration in the hippocampus has been observed (Schmued and Bowyer, 1997) as well as impairment of long-term potentiation in hippocampal-prefrontal cortex pathway (Ishikawa et al., 2005). Sequential learning on a radial arm maze task along with changes in basal gang lia neurochemistry were observed following a toxic dose of METH in rats (Chapman et al., 2001). Human Data A recent report demonstrated lasting depleti ons in prefrontal grey matter density using magnetic resonance imaging (MRI) and impairment in frontal executive f unction even after more than 6 months of abstinence from METH abuse (Kim et al., 2006). Anot her report using positron emission tomography (PET) showed a loss of dopa mine transporters (DAT) in the striatum which was associated with impairments of me mory and motor function (Volkow et al., 2001a). This same group showed a partial recovery of DAT following at least a y ear of abstinence, but there was no significant recove ry of memory and motor func tion (Volkow et al., 2001b). An

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21 earlier study showed lasting de pletions in striatal DAT follo wing an average of 3 years of abstinence (McCann et al., 1998). This leads to the question of whet her the loss of DAT represents an adaptive down-regulation or nerv e terminal damage, which may be long-term. Although some evidence suggests ir reversible damage, the discre pancies in findings could be explained by length of abstinence compared to du ration and extent of use. Using subjects with a range of abstinence from months to years, while at the same time these subjects have use patterns ranging from years to decades, one could reasonably infer that effects of longer abstinence times may be hidden or reduced. Confounding factor s in the human populations sampled may make interpretation less than certain. In support of this theory, however studies in non-human primates indicate a significant recovery in dopamine function following an acute dose of METH only after a prolonged period (Melega et al., 1997; Harvey et al., 2000). These effects are not limited to DAT, a recent study showed significantly decrease d serotonin transporter density in currently abstinent METH abusers associat ed with elevated levels of aggression (Sekine et al., 2006). There is ample evidence that AMPH is neur otoxic to humans, especially the dopaminergic system, however data for whether MDMA is neurot oxic to humans is contr oversial. It has been stipulated that thus far ther e have been no differences found between control subjects and MDMA users in studies using PET, MRI, or EEG (Gouzoulis-Mayfrank and Daumann, 2006). In contrast, in imaging studies, th ere appears to be lasting deficits in the SERT (Buchert et al., 2003; McCann et al., 2005). Results from McCann we re obtained from abstinent human users of MDMA using PET to image the serotonin reuptake transporter (SERT). In this study, levels of SERT were decreased compared to controls wi th a relatively short abstinence period (the requirement being only 2 weeks, and an average of 4 months). However, this reports results were later critiqued as being due to an effect of reduced binding potential rather than reduced

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22 SERT availability by one of the same groups who co-authored the or iginal report (Buchert et al., 2007). There are reports of differences in meta bolic function in some regions such as the hippocampus and amygdala in ecstasy users comp ared to controls, indicating altered brain activity (Obrocki et al., 1999). Some of the problems with human drug abuse da ta in general include: often there is more than one drug being abused, reported usage ma y not be accurate, and investigators dont know what levels (baseline) were there prior to drug use. Also, purity of the tablet cannot be established, especially in th e 1990s, where MDA (3,4-methylenedioxyamphetamine) or MDEA (3,4-methylenedioxyethylamphetamine) were comm only detected in ecstasy pills. A typical tablet of ecstasy is reported to contain 50-150 mg of MDMA (Parrott 200 4). Parrotts study also said that tablets were fairly pure in the 1980s, but that purity then decreased in the mid-1990s, and it has since increased. However, in a st udy by Tanner-Smith (2006), pills were submitted anonymously between 1999-2005, and it appears th at the purity has de creased from 1999-2005. In fact, it was just recently reported that MDMA coming out of Canada had high concentrations of METH, which is a known neurotoxicant (ONDCP, 2008). Addi ng to the controversy over MDMA are the conflicting results obtained from different groups Some studies show deficits just from polydrug use regardless of whether MDMA was involved. This would suggest that it is the combinations of the effects of different drugs that make it neurotoxic. Finally, in human studies we frequently have the problem of non-random assortment of secondary variables (endogenous or exogenous) that might have contribut ed to the subjects a buse patters, and/or inclusion in the study. While there is yet to be direct, conclusive evidence of cellular toxicity/structural damage in humans, there is much evidence of the effect s of MDMA on behavior Regular ecstasy users

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23 demonstrate impaired memory compared to cont rols (Parrott and Lasky, 1998) and also when compared to other polydrug users who had neve r used ecstasy (Morgan, 1999). Regular use has also been reported to result in psychosis, de pression, anxiety, and impulsivity. Some have psychiatric illness in the famil y, suggesting that they may have been predisposed (see above). However, in a prospective study, MDMA users disp layed more psychiatric symptoms than other drug users or normal controls, indicating a direct role for MDMA (Parro t, 2006). The possibility that users of MDMA already had psychiatric symptoms and had not sought treatment, that they are at an increased risk, or that use of other combinations of drugs are confounding factors cannot be ruled out. Therefore, there is no conclusive evidence for a direct involvement of MDMA. Yet there is, however, evidence that MD MA causes oxidative stress in humans. In a study of 120 MDMA users (who had been using for at least one month), levels of lipoperoxidase were increased and levels of Vitamins C and E, -carotene, SOD, and catalase were decreased in the MDMA group compared to controls (Zhou et al., 2003). Acutely, AMPH is a stimulant and results in increased alertness, decreased fatigue, elevated blood pressure, and increased metabolic ra te. Low doses are used therapeutically to treat a wide variety of conditions. It is widely used to treat attention deficit hyperactivity disorder (ADHD) due to its ability to increase focus for i ndividuals with this condition (Faraone, 2007). It has also been used to treat obesity due to its effects on metabolic rate (Bray and Greenway, 1999), and narcolepsy (Soldatos et al., 1979). Chr onic AMPH can lead to deficits in attention, memory, and decision-making skills in humans. Longterm use can also result in what has been termed amphetamine-induced psychosis (Kokkinidi s and Anisman, 1981), typically consisting of paranoia, hallucinations, and beha vioral disorganization. Treatment with antipsychotics generally ameliorates this condition (Leelahanaj et al., 200 5). Impairment of neuropsychological functions

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24 including frontal executive func tion, memory recall impairment, and attention are observed in human chronic METH abusers (Volkow et al ., 2001a; Sim et al., 2002; Kim et al., 2006). In Vitro and In Vivo Models Most in vitro models consist of usin g cell cultures containing monoaminergic neurons or synaptosome preparations. These cultures prov ide an easily controlled environment where variables can be manipulated a nd effects easily observed. Dose s used are generally between 1 and 2 mM, although lower doses can be used, gene rally with limited observable effects (Deng et al., 2002a; Hayat et al., 2006; Warren et al., 2007). These models are typically used to elucidate involvement of a particular biochemical pathway or to evaluate effectiveness of interventions. Slice culture models are also us ed to evaluate effects on specif ic brain regions (Miller et al., 2007). Animal models used depend on the effect bei ng evaluated. For toxicity studies, the two main models used are acute a nd chronic, though the acute toxic dose model is the most common. Acute models are meant to mimic the (binge) use pattern seen in many recreational users. Generally it is either a single acute toxic dose or multiple high doses given within a period of 872 h (McGregor et al., 2003; Itzh ak and Achat-Mendes, 2004). Chr onic models gene rally give a lower dose daily for a period of days to week s (Frederick et al., 1995; Frey et al., 2006). However, there can be substantial differences between the animal models. In mice, MDMA primarily affects the dopaminergic system, while this does not occur in rats and non-human primates. Therefore, it is important to evaluate the advantages and disadvantages of each species employed for experiments.

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25 CHAPTER 2 PLANARIA General Characteristics Planaria are a type of free-living flat worm of the phylum platyhelminthes (class turbellaria) that are believed to possess the first true CNS and bilateral symmetry. They may be similar to the ancestors of all other b ilaterians. They are found throughout the world in freshwater streams and lakes and are well known for their immense regene rative capabilities; thus their popularity in school biology classes. It takes approximately 10,000 (Sanchez Alvarado, 2004) cells to regenerate an entire worm regardless of whence they come. Studies on evolutionary origin of the br ain suggest similar platyhelminthes are the common ancestor for a centralized nervous system in all other verteb rates and invertebrates (Sarnat and Netsky, 2002; Mineta et al., 2003). Thus, they are usually phyl ogenetically located at th e base of the bilateria in the protostome subdivision, alt hough their precise locat ion in such evolution is not completely established. The worms are triploblastic, meaning they ha ve three layers: an ectoderm, endoderm, and mesoderm. They do not contain a body cavity (acoelomate) and th e digestive tract has only one opening for ingestion and elimination. However, they still have complex organ systems. The method of reproduction depends on the species, with some species exhibiting fissionary reproduction and others exhibi ting hermaphroditic sexual repr oduction, or both, which usually depends on the season. Sexually reproducing animals are hermaphroditic, while asexual animals undergo transverse fission to re produce. They do not possess a circ ulatory or respiratory system and therefore must rely on simple diffusion; this may account for their flat shape. The digestive system has a mouth (the opening) which leads to the pharynx and culminates in the branched gastrovascular cavity. One branch extends anteriorly into the head region and two branches

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26 extend posteriorly into the tail section. The excretory system is comprised of flame cells (protonephridia) which have fast-b eating cilia that function to prom ote movement of fluids out of the body through porous openings on the surface (Ishi i, 1980). The protonephridial system is a highly connected, branched system located in all parts of the body. Parenchyma (the mesenchyme) fills the space between organ systems. Planarian movement is largely under cepha lic control (Kato et al., 2004), and they demonstrate little movement in the absence of a head. They exhibit two types of movement: via cilia, which cover the ventral surface of the body, and via contra ctions of the large muscles (more resembling smooth than striated tissu e), the latter enabli ng faster locomotion. Nervous System As stated previously, flatworms are believed to possess the first true CNS. They have a rudimentary bilobar brain consis ting of cephalic ganglia connect ed by two commissures. This gives rise to two ventral nerve cords, also c onnected by multiple commissures, forming a ladderlike structure extending the leng th of the body. These innervate the muscle fibers and sensory receptors. In early studies of the nervous system, many investigators did not realize the complexity of the nervous system as its fi ne structure cannot be observed with a light microscope. Later studies with the electron microscope revealed a much more complex system with synaptic connections and other features characteristic of higher animals. They possess catecholamine (DA and NE), serotonin, glutam ate, GABA, and opioid systems, mostly confirmed only by pharmacological analysis. The brain is arranged in an inverse U-shape. Planaria possess monopolar neurons, typical of invertebrates, in a ddition to bipolar and multipolar neurons (Koopowitz and Chien, 1975). Ne urons located in the commissures in the ventral nerve cords tend to possess bipolar neuron s, possibly functioning as interneurons (Rieger

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27 et al., 1991). The axons are generally not my elinated (Morita and Best, 1966), although some sheathing has been found in other species of flatworm (Trawicki et al., 1988). Cell bodies contain many neurosecretory granules. Neurons can be characterized morphologically by the presence of dense synaptic vesicl es. The nervous system appears to be secretory in nature, with synapses close together (Cebria, 2007). CNS Systems Present Planaria possess many mammalian-relevant neurotransmitter systems, most characterized only via pharmacological methods. However, re cent advances in molecular biology and the perseverance of a select few laboratories have led to molecu lar evidence for serotonin and dopamine systems. The dopamine system may also have different receptor types, although this has not been characterized by cloning or seque ncing. There are also dopaminergic-cholinergic interactions similar to mammalian systems. Much of the early work investigating planarian nervous system was conducted physiologically, providing little evidence for the types of receptors involved. The availability of selectiv e agonists/antagonists has shed more light on the subject, however, molecular characte rizations need to be conducted. Serotonin Serotonin is a substance found throughout the animal kingdom, in single celled organisms and even some plants (Feldman and Lee, 1985; Johnson et al., 2007). In humans it is found in large quantities in the gastroin testinal tract, in blood componen ts, and in the central nervous system. Some of its many functions include pl aying a role in appeti te, anxiety, depression, aggression, learning and memory, temperature regulation, muscular contraction, cardiovascular function, and endocrine regulation. In planaria, serotonin is pres ent throughout the central and peripheral nervous system, particularly along th e ventral nerve cords (Welsh and Williams, 1970; Reuter et al., 1995), with c onfirmation by HPLC (Fernandes et al., 2003; Umeda et al., 2005).

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28 Knockdown of the gene encoding tryptophan hydroxyl ase in planaria, the rate limiting enzyme in serotonin synthesis, resulted in significant decreas es in serotonin, indicati ng that the synthesis in planarians is by a similar pathway (Nishimura et al., 2007a). It is not cl ear what the function of serotonin has in flatworms, but it appears to ha ve roles in regeneration, movement, and circadian rhythms. When given p-Chlorophenalanine, a serot onin depletor, the ability to glide, a function provided by the cilia on the ventral surface of th e worm, decreases sign ificantly (Kimmel and Carlyon, 1990). Instead, these worms rely on the large muscle movement termed looping in the aforementioned paper. Immunohistochemical studies have shown high concentrations of serotonin in the peripheral nerve plexus located in close proximity to the ventral cilia (Welsh and Williams, 1970) and administration of serotonin to muscle fibers stimulates muscle contraction (Moneypenny et al., 2001). Studies in parasitic fl atworms show a more definitive involvement of serotonin, where it is located in the nerve plexus es innervating muscle cel ls (Marks et al., 1994), and administration of serotonin induces muscle contraction, though whether this is a direct or indirect effect remains to be determined. To da te, two G protein-coupled receptors with sequence homology to the 5-HT1A receptor have been cl oned in planaria (Saitoh et al., 1997). This receptor was also characterized with LSD bi nding (Saitoh et al., 1996) and 8-OH-DPAT, a 5HT1A agonist, administration stimulates adenylat e cyclase activity, providing further evidence for a mammalian-like 5-HT1A recep tor (Creti et al., 1992). In mammal s, serotonin is involved in synchronization of circadian rhyt hms (Marchant et al., 1997). Plan aria exhibit fluctuations in serotonin levels corresponding with a light-dark cy cle, even in the absence of light, although its precise role is not yet determin ed (Itoh and Igarashi, 2000). Its role in regeneration has been studied and will be discussed later (see below).

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29 Catecholamines Catacholamines are also present throughout the animal kingdom and have been found in plants. They are involved in a number of behavior al functions. There is also a substantial amount of evidence for a dopaminergic sy stem, however evidence of an adrenergic system is not as strong. The presence of DA and NE has b een confirmed by HPLC and LCED (liquid chromatography with electrochemical detection) (Algeri et al., 1983). The presence of two different DA receptors has putatively been de termined, but this was only characterized pharmacologically (Algeri et al., 1983; Venturini et al., 1989). According to Venturini (1989), there are two distinct behavior al responses to specific drugs (though at high doses): stimulation of D1 receptors results in screw-like hyperkines ias (SLH), whereas stimulation of D2 receptors results in C-like position (CLP). These effects ar e blocked by the relevant selective antagonists. Administration of both D1 and D2 agonists also leads to increases in cAMP levels which is also blocked by the antagonists, a characteristic that has been demonstrated for D1 receptors in vertebrates, but is atypical for D2 receptors (Venturini et al., 1989; Palladini et al., 1996). An increase in cAMP production has been observed with simultaneous stimulation of cannabinoid (CB1) and D2 receptors in a culture of hamster striatal cells, but this was only in combination (Glass and Felder, 1997). A compound commonl y used to ablate dopamine systems in vertebrates, 6-hydroxydopamine (6OHDA) also appears to destroy the DA system in planaria in a similar fashion, resulting in an absence in m ovement and a marked decrease in catecholamines (Caronti et al., 1999). More recently, a gene en coding tyrosine hydroxylase has been cloned in a planarian species and knockdown of this gene attenuates DA synthesis along with movement controlled by the musculature (Nishimura et al., 2007b). Furthermore, 3-Iodo-L-tyrosine, a tyrosine hydroxylase inhibitor, al so leads to a decrease in dopamine production. However, it also leads to a decrease in serotonin (N ess et al., 1996), too, indicating that it may not be specific for

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30 tyrosine hydroxylase as it was demonstrated that knockdown of the actual ge ne led to a decrease in DA, but not 5-HT (Nishimura et al., 2007b). Ther e also appears to be an interaction with the opioid and cholinergic systems in planaria. The c holinergic system may act as an inhibitor to DA-induced movement, as administration of a ch olinergic antagonist re sults in screw-like hyperkinesia (SLH), a behavior typical of D1 a gonists (Venturini et al., 1989; Palladini et al., 1996; Buttarelli et al., 2000) Acetylcholine Acetylcholine is widely known for its role in the muscular system as well as the autonomic nervous system in mammals. It is also presen t as interneurons and projection neurons in the CNS, where it is believed to play a role in arousal, sleep, and lear ning and memory. The two main receptor classes are the ligand-gated nico tinic receptors and the metabotropic G-protein coupled muscarinic receptors. There is eviden ce for the presence of both receptor types in planaria. Physostigmine, an acetylcholinesterase i nhibitor, causes a characteristic movement, or hypokinesia, called bridge-like position (BLP) (Carol ei et al., 1975), an effe ct that is reversed upon addition of atropine, a muscarinic antagonist (Buttarelli et al., 2000 ). Administration of nicotine produces a different mo tor behavior called walnut pos ition (Buttarelli et al., 2000). It should be noted, however, these studies are somewhat compromised by the very high concentrations of drugs employed, at which they may not be as specific. Recently, cloning revealed a planarian gene with sequence homolog y to nicotininc acetylcholine receptor alpha 7-1 subunit with broad expressi on in the planarian CNS (Cebria et al., 2002). Other neurotransmitters Other neuroactive substances such as GABA (Eriksson and Panula, 1994), glutamate (Rawls et al., 2006a), and nitric oxide (Eri ksson, 1996) have also been found in planaria. Although GABA is an important inhibitory transmitter in vertebrates, its role in planaria has not

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31 been established. Even in mammals, during early development some GABA elements are excitatory (Cherubini et al., 1991) Nitric oxide synthase activity has been shown in the pharynx region and is speculated to play a role in feeding behavior, as it has that function in other invertebrates (Eriksson, 1996). Admi nistration of a nitric oxide s ynthase inhibitor did attenuate cocaine withdrawal in planaria, suggesting that it may be invol ved in the withdrawal pathway (Rawls et al., 2006b). However, its function has not been definitively determined. They also possess a cannabinoid system which has been char acterized pharmacologically (Buttarelli et al., 2002). Regenerative Abilities The regenerative ability of planaria has fascin ated scientists since the 1800s. Planaria are capable of regenerating an en tire worm from as few as 10,000 cells (Sanchez Alvarado, 2004). This is due to the presence of totipotent stem ce lls, called neoblasts, located in the parenchyma. They are the only proliferating cells present in these animals; from them, all others differentiate. These neoblast cells continuously replace aged cells throughout the lifetime of the animal in a homeostatic process, and also form the r egeneration blastema for wound healing and regeneration of body parts. Planar ia appear to use a combination of morphallaxis, characterized by a reorganization or exchange of existing tissues, and epimorphosis, characterized by a significant increase in cellular pr oliferation during regeneration. Th ey also appear to go through a continuous process of morphallaxis due to th eir ability to shrink under conditions of food shortage; presumably the tissue is rendered for metabolic substrat es. The mechanisms controlling cellular proliferation, migr ation, and differentiation are under extensive study. Regeneration is initiated by injury or fission. Th e first step is for the wound to be closed by attaching the epidermis on the dorsal and ventra l surfaces together (Chandebois, 1985). This event is followed by abundant cellular proliferat ion of the neoblasts wh ich migrate to the wound

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32 surface and form the regeneration blastema (Sal o, 2006). This generates the missing parts with correct polarity. The mechanisms governing pattern formation and tissue polarity for pl anarians could shed light on why human nervous systems have such limited regeneration potential. Netrin is a chemotropic protein involved in axon guidance. It acts as either a chemoattractant or a chemorepellant depending on the surface receptor s getting the signal (Serafini et al., 1996). Planarian homologs of netrin are required for proper pattern form ation of the cephalic ganglia as well as the ventral nerve cords (Cebria and Newmark, 2005). New t echnologies have led to even more information on the genes required for regene ration in planaria. Many of these, such as PIWI, wnt, and glycogen synthase kinase-3 display similar functions to vert ebrates (Marsal et al., 2003; Reddien et al., 20 05; Adell et al., 2008). Many other factors are involved in the cont rol of regeneration. Serotonin, dopamine, norepinephrine, acetylcholine, and Substance P have all been implicated. Serotonin is believed to be an important early factor stimulating DNA synthesis (Martelly, 1984). Serotonin has also been implicated in neurogenesis in vertebrates, so this might repr esent a conserved role. In rats, both destruction of serotonerg ic axons with the toxin 5,7dihydroxytryptamine (5,7-DHT) and depletion of serotonin with para chlorophenylalanine (PCPA), a 5-HT synthesis inhibitor, results in a significant reduc tion in the number of bromodeoxyuridin e (BrdU)-labelled neurons in the dentate gyrus (DG) compared to control animal s (Brezun and Daszuta, 2000). BrdU, a thymidine analog, specifically labels dividing cells (Gratzner, 1982). In contra st, increasing serotonin levels in the DG results in an increase in neurogenesis in that region (Malberg et al., 2000). Though the exact mechanism by which serotonin regulates neur ogenesis remains to be elucidated, it clearly plays a prominent role. Substance P admini stration enhances cellu lar proliferation in

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33 regenerating planarians (Salo and Baguna, 1986) and acetylcholine has been shown to have inhibitory effects (Lenicque and Feral, 1976). Negative Phototaxis Planarians display a characteristic negative pho totaxis to light, provi ding an easily studied behavioral endpoint (MacRae, 1964; Dasheiff and Dasheiff, 2002). The response is under complex central control, with the brain as the central processing center. Previous studies in D. dorotocephela revealed an absence of a light response when eyes were removed, confirming the specificity of the response to sensing by the eyes (Arees, 1986). The sensing organs responsible for detecting light are located in the eyespot s on the dorsal surface of the body. The cell types that comprise these eyespots are made up of pigment cells and photoreceptor cells. The photoreceptor cells are bipolar neurons which utili ze a rhabdomere as the photosensing organelle (Takano et al., 2007). Pigment cells form a c up around the photosensitiv e elements of the photoreceptor cells. The cell bodies themselv es are outside of the pigment cups. Relative to Vertebrate Systems Neurons in planarian have many similarities to vertebrates, displaying multipolar shape, dendritic spines with synaptic boutons, a si ngle axon, expression of ve rtebrate-like neural proteins, and relatively low spontaneously genera ted electrical activity. In addition, sequence similarity and evolutionary c onservation of many genes important in vertebrate systems have been documented in planaria. Some of thes e include genes important for axon guidance and neural differentiation such as noggi n, an FGF signaling molecule, and -catenin (Mineta et al., 2003). Also, homeobox genes in planaria show high sequence identity with vertebrate orthologs. In addition, they represent distinct brain regi ons with high correlation to vertebrate genes (Umesono et al., 1999). Expression patterns are complicated by the f act that planarians may be undergoing continuous pattern formation as a resu lt of their ability to shrink and/or grow

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34 depending on the availability of food, water temperat ure, and other factors. This results in the activity of some anterior-posteri or homeobox genes in intact plan aria, an event that does not occur in vertebrates (Orii et al., 1999). An understanding of the mechanisms, controls, and signals used in planarian regene ration could add significant insight into the regeneration process for vertebrates and may help elucidate necessary components for therapies using stem cells in vertebrates and humans. Rationale The purpose of the present set of experiments was to characterize the effects of AMPH and MDMA in planaria as a potential model for am phetamines toxicity. Cell culture can provide a good system for evaluating specific effects on specific systems and biochemical pathways, however it fails to provide a good mode l for the microenvironment present in vivo It fails to account for other mitigating factors. Studies in vivo using more complicated animals such as rodents or even insects, on the other hand, involve so many complicat ed interactions that it is hard to separate out what is important for causing the effects versus what is not. Planaria offer an in betweentheir nervous system is simple enough to be able to evaluate effects, yet still offers the microenvironment. The first step of this study was to determin e whether these drugs were toxic at relevant concentrations. In cell culture, dos es used are often in the mM ra nge. We used endpoints of head loss, death, and locomotor speed. The next step wa s to determine if loss of heads in AMPH or MDMA resulted in long-lasting effects following regeneration by looking at locomotor speed over time, along with morphological ch aracteristics. We also wanted to determine if these drugs would have effects on regeneration of heads wh en present during the regeneration process as measured morphologically and by effects on locomotor speed. In addition, we tried

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35 pharmacological interventions that have been shown to be protectiv e in some, but not all, animal and cell culture models, along with other dr ugs as comparisons a nd active controls.

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36 CHAPTER 3 METHODS Maintenance of Planarian Colony Planaria ( Dugesia dorotocephala ) were initially obtained fr om Carolina Biological Supply (Burlington, NC) and maintained in an aerated aquarium with standard ized artificial spring water (1 mM MgSO4, 2.7 mM CaCl2, 0.7 mM NaHCO3, 0.08 mM KCl; pH 7.8). The colony was kept under controlled light conditions (lig hts on at 8AM and off at 5PM). They were fed chicken liver once per week and liver was remove d immediately afterwards to prevent bacterial growth. Water in the tank was changed every tw o weeks. Planaria were harvested on the same day once per week. Once harvested, animals were ke pt in stainless steel dishes and fed every 5 days. Water was changed daily. Animals to be used in experiments were starved for 3 days and then placed in 60 mm plastic dishes (Fisher Sc ientific, USA) with appropriate drug or water. Head Loss and Death Head loss and death were expressed as a ra tio of the number of animals with intact, functional heads or the number of animals alive, respectively, over the number of animals at the beginning of the experiment. Data were expressed as the percentage with intact heads and the percentage alive over time. Negative Phototaxis to Light Planaria exhibit an anti-tropism to light, provi ding an easily measured behavioral endpoint to study the effects of compounds on locomotor sp eed. Speed was measured as time to move away from a standard light gradient over 5cm. The light source was a 40 watt fluorescent lamp placed above the track. The track used was compos ed of a white plastic tray with channels (Figure 3-1). Either drug or water was used to fill the channels. The tray was washed in between

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37 drug treatments and rinsed with distilled water. This procedure was used as a measure of motor function, with a decrease in speed indicating decr eased motor function. Only planaria with intact heads were used to measure speed. Values were gr aphed as the time to travel 5cm, in cm/min. A repeated measures two-way ANOVA with Tukey s honest significant diffe rences post-hoc test was used to detect statistically significant (p< 0.05) differences between groups for all locomotor data. Drug Treatment Amphetamine Dose-Response d-amphetamine was purchased from Sigma Chem ical Co. (St Louis, MO). The initial doseresponse was carried out by using doses of 10nM, 100nM, 1 M, 10 M, and 100 M in 10 mL of standardized water, for 3 days. In a subseque nt dose-response experiment, doses of 10, 20, and 30 M were used for a period of 5 days. Drug and water were changed daily. Head loss, death, and locomotor speed were noted daily. Only animal s with intact heads and at least some adverse response to light were used to measure locomotor speed. MDMA Dose-Response 3,4-methylenedioxymethamphetamine was purchas ed from Sigma Chemical Co. (St Louis, MO). The dose-response was carried out by using doses of 10nM, 100nM, 1 M, 10 M, and 100 M in 10 mL of standardized water, for 4 da ys. Drug and water were changed daily. Head loss, death, and locomotor speed were noted dail y. Only animals with intact heads and light response were used to measure locomotor speed. DOI Dose-Response DOI (2,5-dimethoxy-4-iodoamphetamine), an amphetamine which acts primarily as a 5HT2 receptor agonist with no effects on dopamine or serotonin transporters in mammals, was

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38 purchased from Sigma Chemical Co. (St Louis, MO). This provided an active control drug. The dose-response was carried out by using doses of 10nM, 100nM, 1 M, 10 M, and 100 M in 10 mL of standardized water, for 4 days. Drug and water were changed daily. Head loss, death, and locomotor speed were noted daily. Only animals w ith intact heads and light response were used to measure locomotor speed. Methiothepin Methiothepin was purchased from Tocris Bi oscience (Ellisville, MO). In mammals this affects a number of 5-HT receptors, but not 5-HT re uptake sites. Planaria were treated with water or methiothepin (100nM), for 3 days. Drug and wa ter were changed daily. Head loss, death, and locomotor speed were noted daily. Only animals w ith intact heads and light response were used to measure locomotor speed. Regeneration after Amphetamine Planaria were kept in either artific ial water (control) or amphetamine (30 M) for 3 days. On the third day, animals that had lost heads in amphetamine were removed to a new dish and washed 3 times with artificial water for 5 minutes each to remove drug. Animals that had not lost heads were not used. Control animals were placed in another dish where heads were removed as described (see below) and they were subsequen tly placed in new dishes. Animals were then allowed to regenerate in water for 14 days. Li ght response (reaction to a pen light) was checked and recorded daily. Once all animals had rega ined a light response, locomotor speed was measured until Day 14. Regeneration after MDMA Planaria were kept in either artif icial water (control) or MDMA (100 M) for 5 days. On the fifth day, animals that had lost heads in MDMA were removed to a new dish and washed 3

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39 times with artificial wa ter for 5 minutes each to remove dr ug. Animals that had not lost heads were not used. Control animals were placed in another dish where heads were removed as described (see below) and they were subsequen tly placed in new dishes. Animals were then allowed to regenerate in water for 14 days. Li ght response (reaction to a pen light) was checked and recorded daily. Once all animals had rega ined a light response, locomotor speed was measured until Day 12. Figure 3-1. Test apparatus used to determine loco motor speed of anti-tropism to a standard light gradient. Protein Concentration Determination Protein concentrations in th e pellet were determined us ing a bicinchoninic acid (BCA) protein assay (BCA protein assay kit, Pierce Inc., USA) with albumin standards (Peirce, USA). High Performance Liquid Chromatography Planaria were treated with either 10 or 100 M of amphetamine or MDMA. Samples were collected at 1 h, 24 h, and 72 h and placed in 2.0mL plastic tubes. Only animals with intact heads were collected. Excess water was removed with a pipet. Samples were then immediately frozen

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40 on dry ice and stored in the dark at -80 C until they were ready to be used. In preparation for high performance liquid chromatography (HPLC), 1.0mL of a 0.1% perchloric acid solution containing 100 uM EDTA was added. The tubes were then vortexed and sonicated for 10 seconds. A 100 L aliquot of the mixture was placed in a separate tube to be used for protein analysis. Samples were then centrifuged at 1500 0 G for 10 minutes at 4C. Next, supernatant was removed and filtered with a 0.2 m filter and collected in a separate tube. Filtered supernatant was then injected into an ESA Coulochem II HPLC-ECD (ESA, Chelmsford, MA) using an RP-C18 microdialysi s column with a 20-ul inje ction loop to determine the concentrations of neurotransmitters and their metabolites. The mobile phase consisted of an acetonitrile mixture (MD-TM, ESA). Flow rate was set at 0.6 ml/min. Potential settings on the HPLC were E1 at -175 mV, E2 at 250 mV, GC at 350 mV. The peak s were displayed, integrated and stored by means of an ESA 501 Chromatogr aphy Data System (ESA) Standards for DA, 5HT, and DOPAC (Sigma) run the same day were used to identify a nd quantitate peaks. Values for picograms of compound per sample were calculated by plotting area under the peak against concentration of compound for standa rd solutions. Values were then normalized to protein concentration for each respective sample. The values for control animals were averaged and values for treatment groups are expresse d as %control. A two-way ANOVA with Tukeys post-hoc test was used to detect statistically signifi cant (p<0.05) differences between groups for all HPLC data.

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41 Head Removal for Regeneration For head removal, animals were placed in a large glass Petri dish (100cm wide) and most of the water was removed to minimize movement. Heads were removed with a razor blade just behind the auricles (Figure 3-2), which effected the removal of the entire cephalic ganglia. Figure 3-2. Location of head rem oval. Dotted lines represent the po int at which cuts were made. Drug Combinations In order to ascertain possible mechanisms and whether putative protective agents in mammals would also work in this model, different combinations of drugs were used. In order to evaluate whether drug interventions that have b een shown to be protective in cell culture or animal models, we used combinations of so me of these drugs with AMPH. Lithium was purchased from Tocris Bioscience (Ellisville, MO). Nomifensine maleate and 7-nitroindazole were purchased from Sigma Chemical Co. (St Louis, MO). The first combination used was AMPH and Lithium. In this experiment, planaria were treated with either water, AMPH (27 M), Lithium (1mM, 2mM), or a combination of Lith ium and AMPH. Lithium has antioxidant effects (Shao et al., 2005) and is belie ved to be protective against oxidative stress caused by AMPH (Frey et al., 2006); it also ha s stabilizing effects on second messenger systems. The next combination was nomifensine (Nom), a DA reupt ake inhibitor, and AMPH. AMPH doses of 10

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42 and 100 M were used in combination with a dose of 1 M for Nom. The last combination used was 7-NI, a neuronal Nitric oxide synthase inhibitor. Doses of 20 M AMPH and 50 M 7-NI were used. Doses were chosen based upon both reports in the litera ture (Shao et al., 1995; Itzhak and Ali, 1996) and from separa te dose-response experiments conducted with the planaria. Receptor Binding Assays Samples for the assay were prepared by adding whole animals to 5 mL of assay buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl) and vor texing for 2 minutes. Homogenates were then centrifuged at 10000 G for 10 minutes at 4C. S upernatant was removed and the procedure was repeated for a total of 3 times. The resulting pe llet was resuspended in the total assay volume plus 10% assay buffer. In each well 25 uL each of nonradioactive ligand, radioactive ligand, and sample were combined with 175 uL of assay bu ffer and incubated for 90 minutes. The filter was washed in 0.9% saline to stop the reaction and the plate was filt ered through a GF/B filter strip. The filter was allowed to dry overnight, and scintillation fluid was added to get DPM Binding assays were conducted to determine receptor inter actions and to estimate doses and are reported as dissociation cons tants (Table 4-2).

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43 CHAPTER 4 RESULTS Toxicity of Amphetamine and MDMA Head Loss and Death The first step in evaluating toxicity of am phetamines in this model was to establish a general dose-response. The endpoints used were head loss and death. Treatment with both AMPH and MDMA result ed in head loss. However, AMPH has a steep dose-response curve with head lo ss occurring only at the high dose of 100 M by Day 2, in which there is 100% head loss and no head loss at 10 M or any lower doses until Day 3 (Figure 4-1). Amphetamine at 100 M resulted in 100% death by Day 3, with lower doses having no effect (Figure 4-2). In a subse quent experiment to determine e ffects of intermediate doses, a range of 10 M, 20 M, and 30 M was used. This range produced a greater distribution, with the most head loss occurring at 30 M (100% by Day 4) followed by 20 M (79% by Day 5) and then 10 M (29% by Day 5) (Figure 4-4). Only the high est dose (30mM) resulted in any death (20%) (Figure 4-5). Following treatment with MDMA maximal head loss of 65% occurs at 100 M, with 25% occurring at 10 M and 10% at 1 M (Figure 4-7). Lower doses resulted in no head loss. MDMA did not cause death by Day 4 at any dose (data not shown). Locomotor Speed As mentioned above, dopamine is known to in fluence locomotor movement in planaria (Palladini et al., 1996). Amphetami nes are also known to have effects on the dopamine system, therefore locomotor speed was inco rporated as an endpoint for meas uring toxicity. Control levels were typically between 12 and 14 cm/min. Amph etamine caused decreased locomotor speed at 20 and 30 M for the observed period (p <0.001) (Figure 4-6), and at 100 M, where most animals did not move (Figure 4-3). Locomotor speed was only measured for 3 days with 30 M

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44 AMPH since all worms had lost heads by Day 4. At 20 M, the largest decreases were observed on Days 4 and 5. Locomotor speed was also significantly decreased during administration of 100 M MDMA (p<0.001), but not for any other dose (Figure 4-8). As observed in earlier experiments with AMPH, control values re mained between 12 and 14 cm/min. MDMA (100 M) resulted in observable decreases starting on Da y 1 and speed remained decreased throughout the experiment. The slight increase observed on Day 4 was most likely due to animals that were excluded due to head loss. Morphology Head loss was examined using gross morphologi cal effects as the metric. Planaria were treated with either water or drug. Concentrations of AMPH and MDMA causing maximal numbers of animal with complete head loss w ithout death were used. Following treatment with both (independently) AMPH (30 M) and MDMA (100 M), the tissue on the head appears to be degrading over time (Figure 4-9). Initially, th e auricles, where the chemoreceptors are located, disappear (Figure 4-9B). Following this event, the anterior portion of the head begins to disappear and has the appearance of lesions on th e ventral surface (Figure 4-9B). Lastly, the head appears to be hollow and disintegrates (Figur e 4-9C), leaving a ragged wound surface posterior to where the head used to be (Figure 4-9D). On ly the head tissue is affected by the drugs; the bodies remain unaffected. This same pattern of ev ents is observed afte r treatment with MDMA (100 M) (Figure 4-10A-C). Effects on Monoamines In cell culture and animal models, MDMA and AMPH initially cause an acute increase in serotonin and dopamine release, which is follo wed by substantial depletions. In order to determine if these effects would be obtained in planaria, monoamine levels were measured

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45 following drug administration. AMPH caused signif icant decreases in serotonin levels at 24h with both the 10 and 100 M dose (46% and 62% below contro l, respectively) (Figure 4-11). MDMA (100 M) resulted in decreased serotonin levels at 24h ( 51% below control) and 72 h (33% below control) (Figure 411). While not significant, MDMA at both doses appears to cause an increase in serotonin levels at 1h. MDMA (10 M and 100 M) also resulted in significant increases in dopamine at 1h (279% and 214% over control, respectivel y), with levels returning to normal by 24h and remaining stable at 72h (Figure 4-12). Amph (100 M) caused decreases in DA (39% below control) at 24h (figure 4-12), while 10 M resulted in no significant changes at any timepoint (Figure 4-12). DA turnover, as m easured by calculating th e ratio of DOPAC/DA, was decreased at 1h following administration of both 10 and 100 M MDMA (33% and 51% below control, respectively, p<0.05) (Figure 4-13). For 100 M MDMA, the levels remain depressed at 24h (51% below control, not si gnificant) before jump ing up at 72h (261% over control, p<0.05). AMPH at 100 M also induced increases in DA turnover at 1 and 24 h (140% and 210% over control, respec tively, p<0.05) (Figure 4-13). AMPH Has Long-Term Effects after Regeneration Following head loss due to AMPH, it was thought that there might be persistent effects once the animals regenerated. This was measured by evaluating recovery of locomotor speed. Indeed, planaria that had been treated with AMPH (30 M) were significan tly (p<0.05) slower than control animals, lasting 2 weeks after head loss (F igure 4-14). In contrast, when heads were mechanically removed prior to disintegration in AMPH (30 M), the effects were not significant (Figure 4-17). Worms that lost heads were sl ower than controls by approximately 4 cm/min.

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46 Morphology of Heads Regenerated after AMPH Gross morphology of heads was evaluated to de termine if there were differences after regeneration. Representative phot ographs depict the appearance of heads on Day 14 after they have regenerated (Figure 4-15). Morphology of the heads of some animals that lost heads in AMPH (Figure 4-15B) and subsequently regenera ted them display slightly different gross morphology. The anterior portion of the head re gion in these animals appears blunted. The auricles and eyespots appear normal, however. Not all animals display such gross differences in appearance, however; some appear morphol ogically normal. Animals that had heads mechanically removed do not appear to be di fferent from controls morphologically once heads are regenerated (Figure 4-15C). Loss of Heads in MDMA Does Not Effect He ad Regeneration or Subsequent Locomotor Speed Since loss of heads in AMPH had drastic effects on regeneration, as evaluated with locomotor speed, the effect of losing heads in MDMA was subsequently evaluated. It was found that loss of heads in 100 M MDMA did not effect locomotor speed at any timepoint tested (Figure 4-16). Further, if heads were cut rather than allowed to disinteg rate, there were still no effects on locomotor speed as compared to controls (data not shown). Other Drugs DOI Toxicity DOI, another amphetamine, which acts primarily as a 5-HT2 receptor agonist is a more potent toxin than either MDMA or AMPH and results in 100% head loss after only one day at 100 M and 60% head loss after 4 days at 10 M (Figure 4-18). A dose of 100 M also results in 100% death after only two days (Figure 4-19). He ad loss prevented measuring locomotor speed for the high dose of DOI, but a dose of 10 M resulted in a significant decrease in locomotor

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47 speed as compared to controls (Figure 4-20). Control le vels were slightly lower than what had been previously observed and were between 10 and 12 cm/min. However, locomotor speed of animals that had been treated with DOI (10 M) began to decline on Da y 1 and stayed depressed until Day 4, where the average speed was around 6 cm/min, approximately 5cm/min slower than controls. Methiothepin Potently Inhibits Movement To further compare results, methiothepin, a ge neral serotonin antagonist was administered to planaria. It resulted in dras tic decreases in locomotor speed starting on Day 1 and continuing to Day 2 (Figure 4-21) Control animals started in the range typically observed (between 12 and 14 cm/min) and did decrease slightly to 11 cm/m in. Animals treated with methiothepin on the other hand began at control values and decreased to 5 and less than 3 cm/min on Days 1 and 2, respectively. While the effects on speed were quite pronounce d, there was no effect on head loss or death (data not shown). Nomifensine, Lithium, and 7-Nitroindazole Are Not Protective of AMPH Effects In cell culture and animal models, nomifensine, lithium, amd 7-nitroindazole have all been shown to be protective. We wanted to determine if these same agents would be protective in this model. None of the drug combinations used pr otected against amphetami ne-induced toxicity as defined by head loss and death (Table 4-1). Th e higher dose of Lithium (2mM) appeared to exacerbate the effects, while the lower dose of lithium (1mM), nomifensine, and 7-nitroindazole had no effect on the endpoints measured. Low Doses of AMPH and MDMA Do Not Aff ect Regeneration after Mechanical Removal Serotonin, dopamine, and norepinephrine have been shown to be important players in regeneration (Lenique, 1973). Since AMPH a nd MDMA both affect these neurotransmitter systems, we wanted to ascertain the effects of these compounds on planarian regeneration. Since

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48 high doses proved to be toxic as demonstrated in n earlier experiments, doses in the lower range were chosen. Administration of low doses of AMPH (5 M) (Figure 4-22) and MDMA (10nM, 100nM, 1 M) (Figure 4-23) do not inhibit regenerati on as measured by an index of locomotor speed. Although not significant, planaria treated with MDMA (1 M) perform slightly better than controls. Neither AMPH nor MDMA caused head loss, death, or locomotor speed at these dosage levels (Figures 4-1 to 43 and 4-7 to 4-9, respectively).

57 Figure 4-9. Representative photogr aph of degeneration in head region following exposure to AMPH. In AMPH treated animals (B-D), the auricles begin to di sappear and lesions on the head become apparent (B). Lastl y, the head appears to be hollow (C) and disintegrates (D), leaving a ragged wound surf ace posterior to where the head used to be. Normal worms have a triangular head and intact auricles (A). A C B D

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58 Figure 4-10. Representative photograph of dege neration in head region following exposure to MDMA. In MDMA treated animal s (B-D), the auricles begi n to disappear and lesions on the head become apparent (B). Lastl y, the head appears to be hollow (C) and disintegrates (D), leaving a ragged wound surf ace posterior to where the head used to be. Normal worms have a triangular head and intact auricles (A). A C B D

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59 Figure 4-11. Changes in levels of 5-HT fo llowing treatment with AMPH and MDMA. Levels are reduced at 24 h following AMPH and re turn to normal at 72 h for the low dose (the high dose resulted in complete head loss by 72 h and therefore could not be measured). MDMA induced a decrease at 24 and 72 h with the high dose and at 72 h with the low dose. Values are expresse d as % Control, N=6 samples/group, 3 animals/sample. *p<0.05. 0 20 40 60 80 100 120 140 160 180 200 1h24h72h% Control 10micM AMPH 100micM AMPH 10micM MDMA 100micM MDMA* *

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60 Figure 4-12. Changes in levels of DA followi ng treatment with AMPH and MDMA. Levels are reduced at 24 h following the high dose of AMPH (the high dose resulted in complete head loss by 72 h and therefore could not be measured). MDMA induced an increase at 1 h for both doses with levels returning to normal at 24 and 72 h. N=6 samples/group, 3 animals/sample. *p<0.05. 0 50 100 150 200 250 300 350 1h24h72h% Control 10micM AMPH 100micM AMPH 10micM MDMA 100micM MDMA* *

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61 Figure 4-13. Changes in levels of DOPAC/DA following treatment with AMPH and MDMA. Data are presented as mean S.D. of the % of control values. Levels are reduced at 24 h following the high dose of AMPH (the hi gh dose resulted in complete head loss by 72 h and therefore could not be measured). MDMA induced an increase at 1 h for both doses with levels returning to no rmal at 24 and 72 h. N=6 samples/group, 3 animals/sample. *p<0.05 0 50 100 150 200 250 300 350 400 450 1h24h72h% Control 10micM AMPH 100micM AMPH 10micM MDMA 100micM MDMA* * *

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62 0 5 10 15 20 25Day 0Day8Day10Day12Day14speed (cm/min) Control AMPH Figure 4-14. Head loss following amphetamine expos ure results in long-term locomotor deficits. Planaria that lost heads following ampheta mine exposure and allowed to recover in water fail to return to control levels afte r 14 days. Data are expressed as the mean S.D. of the time to travel 5cm in cm/min. (N=24). Figure 4-15. Representative photographs of h ead morphology after AMPH. Worms treated with AMPH (30 M) and had heads mechanically rem oved (C) did not a ppear different than normal controls (A) after regeneration. Worms that had lost heads in AMPH and regenerated appear slightly blunted at the anterior portion of the head(B). A B C

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63 0 2 4 6 8 10 12 14 16 18 Day 0Day 8Day 10Day 12Speed (cm/min) Control 100 micM Figure 4-16. Head loss following MDMA exposur e does not result in long-term locomotor deficits. Planaria that lo st heads following MDMA exposure and allowed to recover in water for 12 days were not significantly different from controls. Data are expressed as the mean S.D. of the time to travel 5cm in cm/min. (N=24).

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64 0 2 4 6 8 10 12 14 16 Day 0Day 8Day 10Day 12Day 14Speed (cm/min) Control Cut Lost Figure 4-17. Differential effects of losing head s in AMPH vs. being mechanically removed. Consistent with previous observations, animals that lost heads (Lost) following AMPH treatment (30 M) were significantly slower than control animals (p<0.05). Removal of heads (Cut) before head loss occurs does not resu lt in a significant difference from control. Data are expressed as the mean S.D. of the time to travel 5cm in cm/min. (N=24).

70 0 2 4 6 8 10 12 14 16 18Day 0Day 8Day 10Day 12Speed (cm/min) Control AMPH 5micM Figure 4-22. Amphetamine did no t inhibit head regeneration. H ead regeneration was evaluated by measuring locomotor speed which wa s not affected by the presence of amphetamine at any point during regeneration. Data are expressed as the mean S.D. of the time to travel 5cm in cm/min. (N=10/group)

72 CHAPTER 5 DISCUSSION This study sought to characterize the effects of amphetamines in a model using the freeliving freshwater planarian species Dugesia dorotocephala Animal models are time-consuming and costly. Cell culture models allow for investigation of specific effects and are more easily manipulated, but fail to represent the complexitie s found in the CNS. The goal of these studies was to characterize the effect s of AMPH and MDMA in a ne w model employing planaria in order to determine the potential of this inverteb rate for use in drug abus e neurotoxicity studies. Planarians are useful for this type of st udy because of the simplicity of their nervous system and the existing literature on the behavi oral effects of dopaminergic and serotonergic drugs, as well as others (Venturini et al., 1989; Buttarelli et al., 2000). In addition, colonies are easy to maintain and they are very inexpensive. Planarians are already being used as a model system for Parkinsons disease (Kitamura et al., 1998). In this system, MPTP and rotenone tr eatment cause whole body autolysis which is inhibited by typical anti-Parkinsoni an drugs such as talipexole and pramipexole (Kitamira et al., 2003). Planarians have also been suggested as a model system for tumorigenesis studies as well as for environmental toxins (Hansen et al., 1993 ; Schaeffer, 1993). In addition, planarians are being used as a model for drug withdrawal and comb inatorial effects of drugs of abuse (Raffa et al., 2006). This same group has also shown a withdr awal effect from AMPH, using a measure of locomotor velocity, which is inhibited by an opioi d antagonist (Raffa et al ., 2008). In contrast to the measure of locomotor speed we used, this gr oup used a measure of th e number of gridlines crossed in a particular time period under static light conditio ns. Although AMPH has been purported to inhibit regeneration in planaria (Liotti, 1961), there ha ve been no studies to date of which our group is aware investigating the ne urotoxicity of amphetamines in planaria.

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73 Initial Toxicity Studies We initially found that AMPH and MDMA i nduce head loss in a dose x time-dependent manner, with the highest doses exhibiting maximal head loss. The ability of drugs to cause head loss in planaria has not been re ported previously and as such is a novel finding. AMPH appears to have a steep dose-response curve, with th e window of effect being between 30 and 100 M. At 30 M significant head loss is i nduced, but does not result in as much death as at 100 M. The fact that AMPH and MDMA do this in a dose-depe ndent manner suggests that it is a direct effect of the drug. As only heads are affected, it also suggests a unique vulnera bility in the cephalic ganglia. Osmotic disregulation is not likely because the excr etory system responsible for maintainence of osmotic balance, the protone phridial system, is located throughout the body of the worm (Ishii, 1980). If AMPH and MDMA were simply disrupting the equilibrium, then it would be expected to affect the whole body. As the highest dosage levels were also show n to cause reductions in serotonin, which is involved in regeneration, it coul d be interfering with general tissue homeostasis and cellular repair. However, these are speculative and the exact mechanism by which this occurs remains to be elucidated. Morphological examination of he ads that were treated with AMPH and MDMA revealed a gradual autolysis. This could be due to a number of factors, including apoptosis or non-specific necrosis. In worms treated with MPTP and rotenone, whole body autolysis was exhibited which could be inhibited with caspase inhibitors (Kitamura et al., 2003). Proteinases, such as caspase as well as aspartic proteina ses, are present in plan aria and are active in remodeling and regeneration (Zam ora-Veyl et al., 2002; Hwang et al., 2004). Apoptosis has been observed in cell culture and in rodent brain after AMPH and M ETH administration (Cubells et

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74 al., 1994; Cunha-Oliveira et at., 2006; Zhu et al., 2006). Biochemical analysis of caspase levels or levels of apoptosis could help elucidate these effects. Additionally, AMPH and MDMA caused a dose dependent decrease in locomotor speed which also correlated with doses causing head lo ss and death. Earlier st udies indicated that amphetamine behaviorally acts as an indirect ag onist at both the D1 and D2-like receptor types in planaria, resulting in both C-like position (C LP) and screw-like hyperkinesia (SLH) (Venturini et al., 1989). Both DA and serotonin have been implicated in movement control. DA appears to modulate the large muscular cont ractions (Nishimura et al., 2007b) whereas serotonin is thought to modulate ciliary movement (Kimmel and Ca rlyon, 1990). The decreased speed could be due to decreased availability of one or both of thes e neurotransmitters, although other effects such as decreased energy availability might also be possible. The decrease in locomotor speed observed at 24 h following administration of AMPH (100 M) (Figure 4-3) appears to correlate with the decrease in both 5-HT and DA at 24 h, as measured by HPLC (Figures 4-9 and 4-10, respec tively). Acutely, MDMA increases both 5-HT and DA, although this effect is only significan t for DA (Figures 4-9 and 4-10, respectively). MDMA at a dose of 100 M resulted in significant decreases in levels of 5-HT at 24 h that remained depressed at 72 h. The decreases in le vels of serotonin following MDMA treatment are consistent with data in the literature since MD MA acts primarily at the serotonergic system. The increased rate of DA turnover observed with AM PH and MDMA appears to correlate with the timepoint at which maximal head loss occurs for each drug. DA turnover is indicative of an increase in neurotransmitter release which could lead to an increase in oxidative stress. This is an interesting effect since AMPH doe s not increase levels of DA at 1h, but it appears to inhibit the metabolism since the turnover is mu ch higher than in controls. This is in agreement with current

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75 data that AMPH increases DA turnover while d ecreasing the levels of DA, possibly due to inhibition of monoamine oxidase (Pereira et al., 2006). Another amphetamine, DOI, which acts primarily as a 5-HT2 receptor agonist was used as a comparison. This compound proved to be the most potent toxin examined here, with the highest dose of 100 M inducing head loss and death in 100% of animals by Day 1 and Day 2, respectively. A lower dose of 10 M also resulted in 60% head loss by Day 4 and significant decreases in locomotor speed as compared to controls. In humans, DOI is a hallucinogen and exer ts complex actions, di srupting the balance between inhibitory and excitatory influences (Martin-Ruiz et al., 2001). The toxicity caused by DOI in planarians does not occur in rodents. This difference in t oxicity could be partially due to disruption of the serotonin system by DOI, which plays an important role in regeneration and tissue homeostasis in planarians (Franquinet, 1979). Methiothepin, a general seroto nin antagonist, was also used as a comparison to further elucidate results obtained from AMPH and MDMA. It proved to be a potent inhibitor of locomotor movement at a comparatively lo w dose (100nM). The results obtained with methiothepin were not surprising since it has prev iously been shown to be a potent inhibitor of regeneration, although the concen tration used in that experi ment was higher than the concentration used here (500nM and 100nM, respectively) (Sait oh et al., 1996). It has previously been shown that reuptake blockers are protective against neurotoxicity induced by amphetamines (Seiden and Ricuarte, 1987; Malberg et al., 1996). Nomifensine, a DA reuptake blocker, was investigated to determine if it would be pr otective against the toxic effects of amphetamine in this model, as measured by indices of head loss and death. Although it did not have any effect on these parameters, this could be due to a number of issues. Both AMPH and

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76 nomifensine cause C-like position (CLP) and screw-like hyperkinesias (SLH) when given acutely, indicating that they are acting by similar mechanisms (Ven turini et al., 1989). However, it is not known how these two comp ounds interact with the transpor ter or if they would inhibit each other. Another possibility is that the doses used were not sufficient to protect against AMPH toxicity in this model. Evaluating the affinity of nomifen sine with the DAT in planaria using radioligand binding experime nts could help determine not only the best dose range but also could help elucidate the pharmacol ogical activity of this drug in planaria. Further work needs to be done to indicate whether the eff ect of head loss is strictly neuronal. The decreased locomotor speed indicates that there are effects on the DA system, possibly due to oxidative stress or impaired regenerative ability. The possibility that suppor t cells are also being affected cannot be ruled out. Lithium has been shown to have antioxidant effects in mammals (Shao et al., 2005) and is believed to protect against oxidative stress caused by AMPH (Frey et al., 2006). In this model, it exacerbates the eff ects of AMPH. However, Lithium has also been shown to inhibit regeneration in planaria (Lenicque, 1974) and th e result could be due to loss of ability to maintain normal tissue homeostasis, combined with toxicity exerted by AMPH. There is also evidence that reactive nitrog en species play a ro le in toxicity of amphetamines, such as increases in nitrotyrosin e (Darvesh et al., 2005). A neuronal nitric oxide synthase (nNOS) inhibitor, 7-ni troindazole (7-NI), was shown to be protective against METHinduced toxicity in the striatum of mice (Itzhak a nd Ali, 1996). There is also evidence that nitric oxide plays an inhibitory role in muscle move ment (Gustaffson et al., 2001). The inability of 7NI to protect against AMPH-indu ced toxicity, by measures of h ead loss and death, could be due to several factors. 7-NI is a lipophilic com pound and requires dimethylsulfoxide (DMSO) to

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77 dissolve it. DMSO has been shown to be toxic, though at levels much higher than what the animals in this study were exposed to (Pagan et al., 2006). Although vehicl e controls (in DMSO only) did not display toxicity, it is possible that the amount pres ent facilitated uptake of AMPH, or that the two compounds acted in a synergistic way, with AM PH lowering the threshold of resistance to the toxicity of DM SO. Also, since very little is know n about the nitric oxide system in planaria, it may not be interacting in the sa me way it does in mammals. As stated previously, these were conducted as pilot studies and therefor e an exhaustive evaluation of dosages of the putative neuroprotective agents was not conducted. It is possible that these agents might be protective at differe nt dose levels. This model has also been shown to have dopaminergic-cholinergic interactions. The cholinergic system may act as an inhibitor to DA-induced movement, as administration of a cholinergic antagonist results in SLH, a behavior typical of D1 agonists (Venturini et al., 1989; Palladini et al., 1996; Buttarelli et al., 2000). In a rat model, stimulation of the muscarinic acetylcholine receptors inhibited the amphetamine-i nduced increase in DA re lease in the nucleus accumbens (Ichikawa et al., 2002). Administration of an alpha-7 nicotinic acetylcholine receptor (nAChR) antagonist also protected against oxidative stress and toxic ity of MDMA in vitro and in mice brains (Chipana et al., 2008). It would be interesting to examine the effects of cholinergic agonists on the effects of amphetamines in this invertebrate model. Lasting Effects after Head Loss Another interesting finding was th at once planaria lose their heads in AMPH, they fail to recover, as measured by locomotor speed, to contro l levels (Figure 4-13). This would indicate a lasting change. This could be due to lasting depl etions in monoamine levels which subsequently prevent proper regeneration, as they are impli cated in playing a larg e role in regeneration (Franquinet, 1979). More specifically, serotonin is important in the early stages of regeneration

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78 and the rapid increase in DNA synthesis (Mar telly, 1984). In support of the theory that monoamine depletion causes lasting changes, wh en planaria are treated with the serotonin depletor para-chlorophenylalanine, sliced, and allowed to regenera te, they fail to recover normal function (Kimmel and Carlyon, 1990). Thus, it is po ssible that AMPH induces a plastic change in the nervous system which gets perpetuated. Ho wever, the possibility that effects on other components such as tyrosine hydroxylase, which is depleted in mammalian models (Bowyer et al., 1998), cannot be excluded. In this model, AMPH also causes reductions in serotonin levels and therefore could also be a ffecting tryptophan hydroxylase. In addition, since serotonin is thought to be an important modulator of ciliar y movement, while DA is thought to be important for gross muscle movement, it is unclear wh ether there was abnormal development in the musculature or if levels of se rotonin and dopamine simply remain depressed in the regenerates. We did not measure serotonin or dopamine levels in regenerates and therefore do not know what levels were present. The decreased locomotor sp eed appears, in part, to be dependent on AMPH as the causative agent for head loss since head s that were mechanica lly removed were not affected to the same level. These animals did not remain at levels that were significantly below control levels. One explanation for this differential effect could be that animals that did not lose heads also did not have as much monoamine deplet ion, or toxic effect of AMPH in general. In contrast, animals that lost heads in MDMA and were allowed to regenera te were not different from controls. This differential effect is intrigui ng and it could be that toxicity from MDMA is less severe than what occurs with AMPH. The da ta presented here do show that MDMA is less toxic than AMPH on head loss, death, and loco motor speed. It is possible that MDMA is not causing enough of a disruption, or depletion of serotonin and dopami ne, to have lasting effects that are perpetuated after rege neration. Effects on tryptophan hydr oxylase could also participate.

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79 Since AMPH does cause depletions in serotonin in this model, it could also be acting as an inhibitor of tryptophan hydroxylas e. Given the belief that sero tonin is important early in regeneration, a depletion in its levels combined with inhibition of synthesis could lead to the continued effects. Histological studies evaluating the effects on the serotonin and dopamine systems could help elucidate these differential effects. Effect on Regeneration The importance of the monoamines in regene ration led to the hypothe sis that AMPH and MDMA, by altering levels of the monoamines, might impede or inhibit regeneration. In a gerbil model, a single dose of METH temporarily inhibited granule cell proliferation in the hippocampus (Teuchert-Noodt et al., 2000). Sim ilar observations were made following a high binge dose of MDMA in rat brain (Hernndez-Ra baza et al., 2006). In mammals, there are two main sources of neural stem cells in adults. These arise from the sub-granular layer of the hippocampus and the sub-ventricular zone of the lateral ventricles (Zha o et al., 2008) and are speculated to play a role in depression (Eld er et al., 2006) and neur odegenerative diseases (Steiner et al., 2006). In addition, it had prev iously been reported that AMPH inhibited regeneration in planaria (Liotti, 1961). However, this was not the case as evidenced here (Figures 4-17 and 4-18). This disparity is most likely due to a difference in dosages and one possibility is that at low doses these drugs are not producing a level of monoamine depletion that would be inhibitory. However, since higher do ses resulted in toxicity in intact worms, we wanted to test doses that were not toxic by themselves. These lower doses could actually have been helpful by increasing availability of dopamine and serotoni n for regeneration. Although the endpoint used may not be sensitive enough to detect subtle ch anges, there was a sli ght, though not significant, improvement in worms treated with 1 M MDMA. Another, though less li kely, possibility is that

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80 intact heads might be required for the two drugs to exert their effects and th at in the absence of a CNS they fail to modulate monoamine activity. This would further indicate that the toxicity of these two drugs is mediated by the CNS in planarians. Summary and Future Directions In summary, we found that AMPH and MDMA were differentially t oxic in the planarian, with AMPH exerting more toxic effects. AMPH al so displaced 5-HT with high affinity whereas MDMA only bound at micromolar concentrations. Both drugs exerted effects on the serotonin and dopamine systems as evidenced by their effect s on the transmitter levels. We also found that AMPH induces lasting effects on regenerated worms, in contrast to MDMA. Additionally, we found that a 5HT-2 agonist, DOI, was more poten t than either AMPH or MDMA. A general serotonin antagonist, methiothepin, did not cause t oxicity in the form of head loss or death, but had very potent effects on locomotor speed. Thus, the drugs that acted as 5-HT agonists, AMPH and DOI, appeared to exert the most toxicity in terms of head loss and death. Drug intervention used in combination with AMPH did not attenuate effects in this model. It may be that AMPH does not act by the same mechanisms in this system or that the drugs used as protectants are not interacting in the same way, or that different doses were needed. We also found that neither MDMA nor AMPH had effects on regeneration at low doses. The popularity of AMPH and MDMA and the accumulating evidence for lasting neurotoxicity underscores the need for new mode ls to parse out effects. The preliminary evidence of AMPH and MDMA actions in this model suggest it has potential as a model for measuring toxicity of amphetamines and other dr ugs. The simplicity of the nervous system and its similarities to mammalian systems would make planaria an ideal model to segue between in vitro and in vivo systems. However, further studies need to be conducted before it can truly be defined as a legitimate model. The effects need to be confirmed histologically to conclusively

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81 prove that the toxic effects ar e a direct response to administ ration of the amphetamines. The recent advances in planarian and molecular biology should help provide the necessary tools to achieve this goal. Specifically, the cloning of th e serotonin receptor, tr yptophan hydroxylase, and tyrosine hydroxylase genes should help provide for in situ examinations. This is an exciting and potentially great model for quickly evaluating dr ug toxicity. Additionally, the low costs involved could benefit young investigators in a time of fina ncial constraints or for use in smaller side projects that do not have funding.

BIOGRAPHICAL SKETCH Shannon Lee Janssen was born in St. Petersburg, FL in 1978. She attended the Center for Advanced Technologies program at Lakewood High School, graduating in 1996. She then went on to attend the University of Florida obtaining a B.S. degree in chemistry. She had originally intended to go to veterinary school to pursue her love of horses, however she decided this was not the lifestyle she wanted. She then decided to pursue research in the field of drug abuse and addiction and applied to the Un iversity of Florida Interdisciplinary Program in Biomedical Sciences (IDP). She started working with Mark Gold in 1997 and has focused on the neurotoxicity associated with a buse of both legal and illicit drug.